Fabrication Of C-Met Chimeric Antigen Receptor T Cells And Killing Of Hepatocellular Carcinoma Cells In Vitro And In Vivo

Hepatocellular carcinoma(HCC)is one of the most common malignancies in China.Its incidence rate is the third most common malignancy in China,accounting for about 45%of the global incidence of HCC.It has caused serious harm to human life and health.Surgery is the most important treatment for HCC,but HCC is difficult to diagnose in the early stages,in the middle and late stage with distant metastasis and poor prognosis.The prognosis of radiotherapy and chemotherapy is poor,Therefore,immune cell therapy is getting more and more attention.Chimeric antigen receptor(CAR)-modified T cells immunotherapy combines the high affinity of antibodies with the killing ability of T lymphocytes to specifically target antigens that are highly expressed in tumor tissues to kill tumor cells.The CAR-T cell immunotherapy technique has made a breakthrough in the treatment of hematological malignancies,but there was no obvious efficacy in the treatment of solid tumors.Selecting a suitable target antigen is very important for treatment of CAR-T cells.c-Met is a hepatocyte growth factor receptor and is highly expressed in tumor tissues such as hepatocellular carcinoma,breast cancer,nasopharyngeal cancer,lung cancer,gastric cancer,and other tumor tissues.It is expressed at low or no expression in normal tissues and adjacent noncancerous tissues.The high expression of c-Met in HCC through binding to the ligand HGF to activates the HGF/c-Met signaling pathway continuously,resulting in abnormal proliferation of hepatoma cells and inhibiting their apoptosis and increasing their motor and invasive ability.Therefore,c-Met can be a potential target for the treatment of hepatocellular carcinoma.Based on the fully human c-Met Fab antibody previously prepared in our laboratory,by constructing a c-Met CAR lentiviral vector,T-lymphocytes were transfected to produce c-Met CAR-T cells,and detect the killing effect of c-Met-postive hepatocellular carcinoma,to provide a new method for the clinical treatment of liver cancer.METHODS:1.High-affinity and fully human anti-c-Met Fab prepared in the previous laboratory was used as a template to construct anti-c-Met scFv using genetic engineering techniques,and c-Met scFv fragment was incorporated into recombination of lentivirus vectors containing CD8TM,CD28,CD137,CD3ζ,and the second generation c-Met CAR lentivirus vectors were constructed using this as a template.The gene sequencing was used to verify the correctness of the constructed c-Met CAR lentiviral expression vector.The C-Met CAR lentiviral vector plasmid was transfected into X-293T cells with PEI transfection reagent,and its expression in X-293T cells was detected by Western blot.C-Met CAR lentivirus was packaged by virus packaging technology,virus was concentrated using PEG-8000,and virus titers were detected by LaSRT assay.2.To separate peripheral blood by using lymphocyte separation fluid from healthy volunteers,PBMCs in peripheral blood were extracted,PBMCs were activated with anti-CD3 antibody and anti-CD28 antibody,IL-2 was added to promote cell proliferation.The c-Met chimeric antigen receptor was transduced into T lymphocytes through the lentivirus system,and its infection efficiency was detected by flow cytometry.3.To detect and select the hepatoma cell line expressing c-Met by flow cytometry.HepG2 cells were labeled with cell immunofluorescence and co-cultured with c-Met CAR-T cells for 12 h to observe the combinations of CAR-T cells.CCK-8assay was used to detect the killing effect of c-Met CAR-T cells on hepatoma cell lines HepG2 and Bel-7402 in different E:T.Compare the killing effect of shMet-HepG2 in the E:T was 10:1.Establish activated T cells and CD19 CAR-T cells as controls.4.c-Met CAR-T cells were co-cultured with c-Met positive hepatoma cells for24 hours,to collect the culture supernatants,and c-Met CAR-T cells were stimulated by c-Met positive hepatoma cells by ELISA to detect cytokines including IL-2 and IFN-γ.Establish activated T cells and CD19 CAR-T cells as controls.5.Preparation the stable HepG2 cell line labeled with Luciferase.Mouse unilateral armpit tumor formation,and injection of c-Met CAR-T cells adjacent to the tumor,in vivo imaging before each injection to detect c-Met CAR-T cells to detect the inhibition of hepatoma cancer in vivo.Activated T cells and normal saline as controls.RESULTS:1.Successfully constructed the third generation c-Met CAR lentiviral vector plasmid(c-Met-28-137-3ζ),and used it as a template to construct the second generation c-Met CAR lentiviral vector plasmid.The gene sequencing results showed that the plasmid sequence was consistent with the design sequence.The expression of CD3ζwas detected by Western blot.The band size was consistent with the theoretical value.The results showed that the c-Met CAR lentiviral vector plasmid can be effectively expressed in the X-293T cells.Package c-Met CAR lentivirus and concentrated the virus,the virus titer after concentration was approximately 5 x 10~8IFU/ml.2.Infected T cells by lentivirus system.Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was 46.3%~57%.3.The hepatoma cell lines HepG2、Bel-7402 were highly expressing c-Met were successfully selected by flow cytometry.CCK-8 results showed that the E:T was20:1,10:1,5:1,2:1.the killing efficiency of HepG2 cells in the third generation c-Met CAR-T cells was 83.5±2.59%,72.4±2.42%,60.93±2.3%,53.07±3.11%,c-Met CAR-T cells was statistically different from CD19 CAR-T cells and activated T cells(P<0.05).The result is not statistically different from the second-generation c-Met CAR-T cells(P>0.05).When the E:T was 10:1,the cytotoxicity of c-Met CAR-T cells against c-Met low expressing shMet-HepG2 cells was significantly different from that of c-Met highly expressed HepG2 cells(P<0.05),but CD19 CAR-T cells and activated T cells have no statistical difference in the killing ability of HepG2 and shMet-HepG2(P>0.05).4.ELISAresultsshowedthatc-Met-28-137-3ζ,c-Met-137-3ζ,c-Met-28-3ζCAR-T co-cultured with HepG2 cells at an E:T of 10:1 secrete IFN-γin the culture supernatant was 2066.7±251.66 pg/ml,2013.2±202.5 pg/ml,1606.7±275.92 pg/ml.The results showed that the second and third generation c-Met CAR-T cells with co-stimulatory factor CD137 can secrete more IFN-γthan second generation and the first with no co-stimulatory factor CD137.Compared with c-Met in statistically significant difference(P<0.05).The secrete of IL-2 in the culture supernatants were 1496.67±230.29 pg/ml,1113.33±61.1 pg/ml,1273.33±46.19 pg/m.The analysis showed that the IL-2 secreted by the third-generation c-Met CAR-T cells was higher than the second generation c-Met CAR-T cells(P<0.05).The secretion of IFN-γand IL-2 in c-Met CAR-T cells co-cultured with shMet-HepG2 with low c-Met expression was lower than that in c-Met CAR-T cells co-cultured with high c-Metexpression HepG2 cells(P<0.05).5.In vivo imaging results showed that c-Met CAR-T cells can inhibit HepG2cell proliferation in vivo.The inhibitory rates of c-Met-28-137-3ζCAR-T cells,c-Met-137-3ζCAR-T cells、c-Met-28-3ζCAR-T cells and activated T cells in vivo were65.3%、49.8%、43.1%、6.7%.There was a statistically significant difference between the c-Met CAR-T cell group and the control group(P<0.05).The inhibitory effect of the second generation was statistically different from third generation c-Met CAR-T cells(P<0.05).CONCLUSIONS:1.Constructed the third-generation c-Met CAR(c-Met-28-137-3ζ)and second-generation c-Met CAR(c-Met-28-3ζ、c-Met-137-3ζ)lentiviral expression vector and packaged and prepared the c-Met CAR lentivirus.Fabricate the third-generation and second-generation c-Met CAR-T cells.2.The c-Met CAR-T cells can specifically kill c-Met positive hepatoma cells and secrete cytokines IFN-γand IL-2,and as the target cell c-Met expression level decreases,the killing ability and cytokine secretion ability of the c-Met CAR-T cells also decrease.3.C-Met CAR-T cells can effectively inhibit the growth of c-Met-positive liver cancer in vivo,and the third-generation c-Met CAR-T cells inhibit the growth of liver cancer more effectively than the second-generation c-Met CAR-T cells.